Project Summary ROR?t is required for the differentiation of type 3 innate lymphoid cells (ILC3) and T helper 17 cells (Th17) that not only protect barrier surfaces from infection but also contribute significantly to inflammatory diseases. Using proteomics approaches, we found ROR?t to be heavily modified post-translationally (PTM) and interact with a number of protein and RNA coregulators. In addition to the known phosphorylation and acetylation previously documented, we found novel phosphorylation at serine 510 and ubiquitination at lysine 516 of ROR?t. To evaluate the implication of these PTMs, we have generated knock-in mice carrying modification-null alleles at the endogenous rorc locus. ROR?t target gene expressions were significantly reduced in PTM mutant animals in a cell type and tissue specific manner. A more detailed characterization of how ROR?t is regulated by these PTMs and their contribution to tissue-specific ROR?t interaction partners may provide new approaches for therapeutic intervention in the setting of immunity and autoimmune conditions. For the first aim, we will determine the role of ROR?t PTMs in ILC3-mediated protective immunity and Th17-dependent autoimmune conditions. In Aim 2, we will evaluate the contribution of ROR?t PTM null mutations to the genomic occupancy of ROR?t and its transcription coregulators, chromatin accessibility, and global transcriptions in Th17 cells. In Aim 3, we will identify upstream enzymes essential for modifying ROR?t at S510 and K516 in vivo.